Abstract: BACKGROUND: The traditional method for treating infected bone defects is thorough debridement, filling with antibiotic-impregnated bone cement, and then autologous bone transplantation. However, there are problems such as insufficient antibiotic concentration and drug resistance, and limited bone mass. Therefore, it is of great clinical significance to seek dual-functional biomaterials that can locally release antibiotics at the site of infection and promote bone repair.
OBJECTIVE: To design an injectable hydrogel composed of hyaluronic acid and oxidized dextran as a local delivery system for vancomycin to treat infection and promote bone regeneration.
METHODS: (1) Vancomycin-loaded hyaluronic acid/oxidized dextran hydrogels were prepared to characterize the morphology, mechanical properties, and in vitro drug release of the hydrogels. (2) Hyaluronic acid/oxidized dextran hydrogels and vancomycin-loaded hyaluronic acid/oxidized dextran hydrogels were co-cultured with rabbit bone marrow mesenchymal stem cells. Cell activity and proliferation were detected by live-dead staining and CCK-8 assay. After osteogenic induction, alkaline phosphatase staining, alizarin red staining, RUNX2 immunofluorescence staining, and RT-qPCR detection (osteocalcin and bone morphogenetic protein 2 mRNA expression) were performed to evaluate the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. (3) The above two hydrogels were co-cultured with Escherichia coli (or Staphylococcus aureus) to detect the antibacterial ability of the hydrogels. (4) Thirty rabbits were selected to establish a 1.5 cm infected bone defect model in the middle of the left radius. Two weeks after modeling, they were randomly divided into three intervention groups: the blank group (n=10) did not receive any treatment; the control group (n=10) was injected with hyaluronic acid/oxidized dextran hydrogel at the bone defect site; the experimental group (n=10) was injected with vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel at the bone defect site. 12 weeks after injection, the samples were collected for Micro-CT scanning, tissue morphology observation, RT-qPCR detection (tumor necrosis factor α, interleukin 6 mRNA expression), and immunohistochemical staining.
RESULTS AND CONCLUSION: (1) Vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel had a good porous structure with a pore size between 100-300 μm, and had good mechanical properties and in vitro drug sustained release performance. (2) Live-dead staining and CCK-8 assay results confirmed that vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel had good biocompatibility. Alkaline phosphatase staining, alizarin red staining, RUNX2 immunofluorescence staining, and RT-qPCR test results showed that both hydrogels could promote osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. (3) Compared with hyaluronic acid/oxidized dextran hydrogel, vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel could significantly inhibit the growth of Escherichia coli and Staphylococcus aureus. (4) Micro-CT scanning results showed that the bone volume fraction and bone density of new bone in the bone defect area of the experimental group were higher than those of the blank group and the control group (P < 0.05). The bone tissue morphology observation results showed that the experimental group had better repair effect of bone defects compared with the blank group and control group. The expression of tumor necrosis factor α and interleukin 6 mRNA in the bone defect site in the experimental group was lower than that in the blank group and control group (P < 0.05). Immunohistochemical staining showed that the protein expressions of osteocalcin and RUNX2 at the site of bone defects in the experimental group and control group were higher than those in the blank group (P < 0.05). These findings indicate that vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel can effectively promote bone regeneration under infection.

Key words: infected bone defect, drug delivery system, hyaluronic acid, oxidized dextran, hydrogel, vancomycin, bone repair, engineered bone material